A Methodology for the Enzymatic Isolation of Embryonic Hypothalamus Tissue and Its Acute or Post-Culture Analysis by Multiplex Hybridisation Chain Reaction

The hypothalamus is an evolutionarily ancient part of the vertebrate ventral forebrain that integrates the dialogue between environment, peripheral body, and brain to centrally govern an array of physiologies and behaviours. Characterizing the mechanisms that control hypothalamic development illuminates both hypothalamic organization and function. Critical to the ability to unravel such mechanisms is the skill to isolate hypothalamic tissue, enabling both its acute analysis and its analysis after explant and culture. Tissue explants, in which cells develop in a manner analogous to their in vivo counterparts, are a highly effective tool to investigate the extrinsic signals and tissue-intrinsic self-organising features that drive hypothalamic development. The hypothalamus, however, is induced and patterned at neural tube stages of development, when the tissue is difficult to isolate, and its resident cells complex to define. No single molecular marker distinguishes early hypothalamic progenitor subsets from other cell types in the neural tube, and so their accurate dissection requires the simultaneous analysis of multiple proteins or mRNAs, techniques that were previously limited by antibody availability or were arduous to perform. Here, we overcome these challenges. We describe methodologies to precisely isolate early hypothalamic tissue from the embryonic chick at three distinct patterning stages and to culture hypothalamic explants in three-dimensional gels. We then describe optimised protocols for the analysis of embryos, isolated embryonic tissue, or cultured hypothalamic explants by multiplex hybridisation chain reaction. These methods can be applied to other vertebrates, including mouse, and to other tissue types. Key features • Detailed protocols for enzymatic isolation of embryonic chick hypothalamus at three patterning stages; methods can be extended to other vertebrates and tissues. • Brief methodologies for three-dimensional culture of hypothalamic tissue explants. • Optimised protocols for multiplex hybridisation chain reaction for analysis of embryos, isolated embryonic tissues, or explants.


Graphical overview Background
In the developing embryo, dynamic cellular and molecular processes direct early tissue patterning and subsequent organogenesis.These events have been deciphered through multiple experimental approaches, but one of the most indispensable tools in such investigations is the use of tissue or organ explants.While organs-including the central nervous system-can be isolated manually ( (Placzek et al., 1990).This neutral protease can separate intact tissue layers, preserving the viability of each layer, and has frequently been used to isolate the posterior neural tube (Placzek et al., 1990;Yamada et al., 1993;Ericson et al., 1997;Tozer et al., 2013).Explants from isolated posterior neural tube regions, or adjacent tissues, have enabled an exquisite dissection of the cellular interactions and signalling events that orchestrate spinal cord patterning.In combination with post-hoc analysis through immunohistochemistry or in situ hybridisation, their use has helped to reveal how distinct progenitor subtypes are established along the dorso-ventral axis of the prospective spinal cord in response to dynamic morphogen gradients (Grega, 1984 The anterior neural tube that will give rise to the brain is more difficult to isolate due to its small size and dynamic morphogenesis.Nonetheless, where anterior neural tube explants have been used, they, too, have provided new insights into the tissue, cell, and molecular interactions that mediate brain patterning (Ericson et al., 1995;Guinazu et al., 2007;Placzek and Briscoe, 2018;Shirahama et al., 2019).In particular, their use has significantly improved our understanding of the development of the hypothalamus (Dale et al., 1997;Manning et al., 2006;Kim et al., 2022;Chinnaiya et al., 2023).This intricate region of the ventral forebrain regulates physiological processes that maintain homeostasis and orchestrate complex behaviours.The enzymatic isolation of entire embryonic chick hypothalamic tissues at different patterning stages, and their acute analysis through scRNA-seq, has generated a comprehensive roadmap of hypothalamic development, from induction to patterning and to neurogenesis, and revealed many previously uncharacterized candidate regulators of hypothalamic patterning (Kim et al., 2022).A powerful new development has been the use of multiplex hybridisation chain reaction (HCR) (Choi et al., 2018)

A. Removal of embryo from egg
The following section describes the removal of a chick embryo from the egg.The method is applicable to all patterning stages of development.Isolation can be performed outside a laminar flow hood, but eggs and instruments should be sterilised with 70% ethanol and, between use, instruments should be placed on sterilised tissue paper (autoclaving dissection tools is not necessary).1. Incubate embryos in a 37 °C incubator to appropriate HH stage.Use standard fine scissors to window the eggshell and No. 5 fine-tipped forceps to carefully peel away outer and inner shell membranes and vitelline membrane, without damaging the embryo.2. Use No. 5 fine-tipped forceps and standard fine scissors to cut around the embryo.Lift out and place in ice cold L-15 medium in a 10 cm Petri dish (Figure 1B). 3. Remove the chorioallantoic membrane and any contaminating yolk.Transfer embryo (with a minimal amount of medium) to clean L-15 medium in a 30 mm Petri dish using a wide-bore 3 mL Pasteur pipette and assess developmental stage.4. Cut away excess extra-embryonic tissue using Vannas scissors or an electrolytically sharpened tungsten needle, to leave the embryo surrounded by a small amount of extra-embryonic tissue (Figure 1C). 5.If required, analyse a small piece of embryonic tissue by PCR to determine sex.The protocol we describe below is equally applicable to male and female animals.Top tip: 5 min rule.After removal from the egg, isolate the embryo from extraembryonic tissues as rapidly as possible (within 5 min).Transfer embryo to ice and keep on ice at all times to avoid tissue and RNA degradation.

B. Hypothalamic isolation B1. Method 1 Isolation of neuroectoderm
The following section describes the isolation of anterior neuroectoderm at three different stages: neural plate [Hamburger-Hamilton (HH6-HH8)], neural tube (HH9-HH12), and phylogenetic (HH13-HH25) stages.The same protocol can be adapted to isolate tissues from other germ layers and other species.Dispase is particularly effective in rapidly and gently separating the neuroectoderm from other tissue layers, while preserving its integrity and viability.1.Using tungsten needles, trim embryo in cold L-15 medium to isolate anterior embryonic regions, discarding posterior portions of the embryo and any remaining extraembryonic tissue (Figure 2A, 2F, 2K).
Leave sufficient excess tissue to avoid damaging the hypothalamus at subsequent steps in the procedure, so make a posterior cut at the level of Hensen's node (at HH6), somite 1 (at HH7-HH12), and midbrain (HH13-HH25).2. Transfer the anterior embryonic region into 0.5 mL of freshly made Dispase (1 mg/mL) in L-15 medium in a Nunc 4-well dish at room temperature.Depending on the size of tissue, transfer using siliconised glass pipette, appropriately sized Gilson pipette (P200 or P1000), or forceps (from small to large size, respectively), taking care to transfer minimal amounts of L-15 medium.The length of time of incubation is highly stage dependent (see Table 1).A rule of thumb is to observe the embryos until the tissue layers begin to visibly separate: the mesendoderm shrinks relative to ectoderm (Figure 2B, 2G, 2L).Once the reaction is complete, transfer the embryo regions from Dispase solution into a large volume of cold L-15 medium in a 10 cm dish.Allow embryos/embryonic regions to rest for 5-10 min.

Hypothalamus dissection
The following section describes the isolation of hypothalamic tissue at neural plate, neural tube, and phylogenetic stages.
1. Use tungsten needles (some prefer a combination of tungsten needles and micro knives) to dissect the neural tissue (i.e., neural plate, neural tube, or brain, depending on embryonic stage) away from other germ layers.To do so, use a holding needle in one hand to pin down the embryo region (avoid pinning down the hypothalamus), and use the long edge of the second needle to gently stroke away the mesendoderm, paring it from adjoining neuroectoderm.The ease and details through which this is achieved is stage dependent.At neural plate stages, the mesendoderm can be easily stroked away from adjacent neuroectoderm.The tissues can be readily distinguished: neuroectoderm is smoother and larger than mesendoderm (Figure 2C).The anterior-posterior axis can be distinguished through the headfold; the neural plate medial midline (which harbours prospective floor plate and hypothalamus) is obvious through its translucent appearance (Figure 2C, dotted outline).At neural plate stages, the neuroectoderm is contiguous with ectoderm, which will provide a useful holding tissue at subsequent stages of dissection.At neural tube and phylogenetic stages, mesendoderm and neuroectoderm do not separate as easily, and mesendoderm must be carefully peeled away around the optic vesicles, optic stalk and eyes, and hypothalamus.This can be achieved using tungsten needles and (where tissues are larger) sharp No. 5 forceps.Once isolated, the neural tissue should appear smooth and epithelial-like, with no contamination of round mesenchymal/mesendoderm cells (Figure 2H, 2M).Transfer isolated neuroectoderm into cold L15 medium using a siliconised glass Pasteur pipette or an appropriately sized Gilson pipette (P10, P20), again ensuring that the tissue does not rise to meniscus and bursts.2. Once the neural tissue is dissected, sub-dissect the hypothalamus (Figure 2D, 2H, 2I, 2M, 2N) using morphological criteria to define its limits.At HH6, the prospective hypothalamus is situated in and around anterior-most medial midline cells.Like more posterior midline cells, these are translucent but can be distinguished from these because they are slightly wider (Figure 2D).At HH10, the nascent hypothalamus occupies an area centred around a series of neuroepithelial folds in the ventral neuroectoderm.While most obvious after contrast microscopy (e.g., Figure 2I), the folds are morphologically visible under the dissecting microscope (Figure 2H) and provide a precise reference point for hypothalamic position.At later phylogenetic stages, the hypothalamus protrudes ventrally between the morphologically distinct optic chiasm and cephalic flexure (Figure 2L, 2M).Isolation of hypothalamic tissue is achieved in two steps.First, a domain encompassing the optic stalk, hypothalamus, and more dorsal diencephalic tissue is dissected using tungsten needles (limits shown by ellipse in Figure 2M, isolated region shown in Figure 2N).Second, this tissue is prepared as an open book.The hypothalamus is obvious as a violin-shaped domain, composed of a translucent medial domain and a thicker surrounding area, situated posterior to the optic stalks/developing optic chiasm (ellipse in Figure 2O).Top tip 1: 5 min rule.Except for the Dispase step, avoid removing the embryo from ice-cold L-15 medium for more than 5 min.With practice, the neuroectoderm can be isolated from the mesendoderm within 30-60 s (early stages) and 2-3 min (late stages).A good rule of thumb is to dissect one embryo at a time.Top tip 2: At neural plate stages, proceed to dissect out the hypothalamus (Figure 2C) as rapidly as possible, or midline cells lose their translucent appearance.Top tip 3: Routinely sharpen the cutting tungsten needle.Top tip 4: Dissect in a relaxed and calm environment.Remember to breathe and keep your shoulders loose and back straight.

B2. Alternate slicing method
In the previous method, the anterior neural plate/neural tube is first isolated using Dispase, and the hypothalamus (or prospective hypothalamus) is then sub-dissected.Some find this approach difficult and prefer the following alternate method, in which embryos are first sliced into fragments, including a hypothalamiccontaining fragment that is then treated with Dispase, and hypothalamic tissue isolated.This method is particularly useful for neural tube-stage embryos, where some find it difficult to pare away mesendoderm from the neuroepithelial folds that characterise the nascent hypothalamus at this stage.1. Transfer the dissected embryo to a clean 5 cm square plastic plate with as little liquid as possible (it must not dry out but must not float) and arrange it so it is flat (Figure 3A). 2. Set the plate with the embryo onto the base of the tissue chopper, with the anterior-posterior axis of the embryo at a right angle to the tissue chopper blade (Figure 3B). 3. Set the thickness of each slice by turning the thickness dial at the front (Figure 3C) and use a new razor blade for each experiment to keep the sample sterile.4. Move the holder base to the start position and press start.5. Once the tissue is sliced (Figure 3D), carefully transfer anterior slices to L-15 medium in a 30 mm Petri dish using a siliconised glass pipette or Gilson (P2 or P10).Each slice has a distinctive morphology.Slices containing the hypothalamus are thicker than more anterior slices, due to the folded neuroepithelium and the underlying mesendoderm, and have a wider neural tube than more posterior slices (Figure 3E).6. Transfer hypothalamus slices to Dispase for 5 min (Figure 3F); then, isolate the neural tissue by gently stroking away the adjacent mesendoderm, as above.Once freed from adjacent tissue, the neuroepithelium will constrict slightly (Figure 3G, 3G'), but the ventral midline containing the nascent hypothalamus remains distinct and can be sub-dissected with tungsten needles (Figure 3G").Isolated hypothalamic tissue can be subjected to a wide range of further investigations, ranging from tests of function in vivo (e.g., grafting it to ectopic locations or cell dissociation for scRNA-Seq analyses) to tests of function ex vivo (e.g., neurospherogenic competence or ex vivo cultures).Here, we focus on the investigation of hypothalamic tissue explants in 3D culture systems.Tissue explants are a highly effective tool to investigate the extrinsic signals and tissue-intrinsic self-organising features that orchestrate hypothalamus development.They are versatile and can be used to assay patterning, proliferation, and migration.Explant cultures may consist of the entire hypothalamus or smaller, sub-dissected domains (limited by size).Good viability and the development of explants in a manner analogous to their in vivo counterparts occurs when explants are relatively small.Explants of this size can be cultured for up to seven days, after which tissue crowding and cell death begin to be observed in the explant centre.When used to assay the effects of signalling ligands or inhibitors, these factors can be added along with explant medium.Explants can be embedded in different matrices, most frequently in collagen or Matrigel.Explants are placed on a bottom layer of gelled collagen or Matrigel, then covered with a second layer, and positioned at the interface of the layers.Previous reports have provided detailed methodologies for how to embed explants in 3D matrices (Placzek and Dale, 1999;Placzek, 2008), so here we provide only brief details.  3. Transfer explants onto the collagen bed using a siliconised glass pipette/appropriately sized Gilson (P2, P10, P20), position them, and remove excess medium using a fine pipette (Figure 4B).Overlay with 25 μL of collagen, spreading this to ensure it covers the bottom bed.Reposition/manipulate the explants as required while the collagen is setting.Collagen sets quickly at room temperature so work rapidly and add top collagen layer to one well at a time, providing the opportunity to reposition explants before the collagen begins to set. 4. Allow the top collagen bed to set completely at room temperature (check by prodding the collagen with a tungsten needle; set collagen feels firm) before adding 400-500 μL of explant medium with or without factors and transfer to incubator for the desired time of incubation (Figure 4C, 4D).Routinely, we culture explants for 3 h to 7 days.
For Matrigel: 1. Prepare desired volume (25 μL/bed/well) of 50% Matrigel and 50% explant medium in an Eppendorf and vortex for 15-20 s.If unused, Matrigel should be left on ice as it will start to set at room temperature.Always make a fresh batch of Matrigel for bottom and top beds.2. Proceed with steps 2-4 as above for collagen, but beds must be set by placing dish at 37 °C for a minimum of 30 min (check by prodding Matrigel with a tungsten needle; set Matrigel feels firm).

Data analysis
Analysis by multiplex HCR  Embryos and explants can each be processed as wholemounts or as cryosections, as detailed below.After completion of the HCR, neuroectoderm can be particularly well visualised either by hemi-dissecting embryos along a sagittal midline (we refer to these as hemiviews), or by isolating the neuroectoderm.Depending on the stage, this can be Moore and Kennedy, 2008; Morrison et al., 2021), tissue isolation at patterning stages requires an enzymatic treatment.A variety of different enzymes can be used, including Collagenase, Trypsin, Dispase I, or Dispase II (Davies, 2010; Lowery et al., 2012; Morales et al., 2016; Shirahama et al., 2019; Ye et al., 2022), amongst which Dispase I is particularly effective ; Placzek et al., 1990; Yamada et al., 1993; Ericson et al., 1997; Tozer et al., 2013; Morales et al., 2016; Placzek and Briscoe, 2018).

Figure 1 .
Figure 1.Experimental setup for harvesting chick embryos.(A) Required dissection tools.(B) L-15 medium on ice and Brown Bovan fertilised eggs.(C) Examples of HH10 chick embryos after harvesting from the eggs and removal of membranes, yolk, and excess extra-embryonic tissue.Scale bar: 1 mm.
Cite as: Chinnaiya, K. and Placzek, M. (2023).A Methodology for the Enzymatic Isolation of Embryonic Hypothalamus Tissue and Its Acute or Post-Culture Analysis by Multiplex Hybridisation Chain Reaction.Bio-protocol 13

Figure 2 . 7 Published:
Figure 2. Steps involved in Dispase treatment and sub dissection of hypothalamic tissue at neural plate, neural tube, and phylogenetic stages of development.(A, F, K) Ventral views of whole embryos (A, F) or side view of head (K) harvested from eggs before Dispase treatment.At late phylogenetic stages, the eye is removed prior to Dispase treatment.(B-D) Isolation at neural plate stages.(B) Boxed region in (A) following isolation and Dispase treatment.(C) HH6 neuroectoderm or mesendoderm, separated after Dispase treatment.Dotted outline shows translucent medial midline neuroectoderm cells.(D) Same

Figure 3 .
Figure 3. Isolation of hypothalamus tissue using the slicing method.(A) HH9 embryo laid flat on a plastic plate.(B) Top view of tissue chopper, with plastic plate and embryo (dotted outline and arrow) ready for slicing.(C) Front view of tissue chopper.(D) HH9 embryo sliced into 150 μm thick slices.(E) Anterior-most slices, removed from the plastic holder, arranged from anterior (top) to posterior (bottom).Arrows point to hypothalamus-containing slice.(F) Examples of HH10 slices, each containing hypothalamus, after Dispase treatment.(G) Slice containing hypothalamus from a HH9 embryo after Dispase treatment: dotted outline demarcates neuroectoderm and mesendoderm.(G'-G") Same slice after isolation of neuroectoderm (G') and sub-dissection to obtain hypothalamic explant (G").Scale bar: 250 μm.
Cite as: Chinnaiya, K. and Placzek, M. (2023).A Methodology for the Enzymatic Isolation of Embryonic Hypothalamus Tissue and Its Acute or Post-Culture Analysis by Multiplex Hybridisation Chain Reaction.Bio-protocol 13(23): e4898.embedding for three-dimensional explant culture

For collagen: 1 .
Prepare the desired volume (25 μL/bed/well) of 90% collagen and 10% 10× DMEM in an Eppendorf and vortex for 15-20 s.Add 0.8M NaHCO3 to make the solution turn pale yellow after vortexing.Typically, 2-6 μL of NaHCO 3 is added to 100 μL of collagen/10× DMEM.Collagen, if unused, should be left on ice as it will start to set at room temperature.Always make fresh batches of collagen for bottom and top layers.2. In a Nunc 4-well dish, prepare collagen beds by pipetting 20-25 μL of collagen, spreading it into a flatbed (Figure4A).Allow to set (20-30 min at room temperature).Flat collagen beds are easier to embed the tissue as the tissue tends to slide off a convex bed.
Embryos and cultured explants can be analysed by immunohistochemistry(Manning et al., 2006), flow cytometry(Perez et al. 2023), chromogenic in situ hybridisation(Manning et al., 2006), or multiplex hybridisation chain reaction (HCR).In our hands, we have found that immunohistochemistry and chromogenic in situ work equally well for explants embedded in either collagen or Matrigel, but HCR works well only in explants embedded in Matrigel.Previous reports have described methodologies for multiplexed quantitative HCR(Choi et al., 2020) and for its extension to immunohistochemistry(Schwarzkopf et al., 2021).Here, we extend these reports to provide details on how to take embryos and explants through repeated rounds of multiplex HCR.Such stripping and reprobing enables samples to be routinely analysed with 10-12 genes per sample (Chinnaiya et al., 2023) (Figure5).

Figure 5 .
Figure 5. Wholemount multiplex hybridisation chain reaction (HCR) analysis on embryos and explants (A-D) HCR analysis on hemi-dissected HH20.Views show hypothalamic region in four sequential rounds of HCR following stripping and re-probing.Tuberal regions of the hypothalamus express ISL1, SIX6, SHH, FGF10, TBX2, RAX, and NKX2-1; hypothalamic mammillary and supramammillary regions express FOXA2, PITX2, and EMX2.NKX2-1 is additionally expressed in the ventral telencephalon and PAX6 in the dorsal diencephalon (from (Chinnaiya et al., 2023).(B) Multiplex HCR analyses on wholemount HH6 explants embedded in Matrigel and cultured for 72 h.The explants have been taken through four rounds of HCR following stripping and re-probing.In this example, the explant included prospective tuberal hypothalamus and some eye tissue.Scale bar: 100 μm.

Table 1 . Duration of Dispase treatment for the different chick developmental stages HH stage Duration of 1 mg/mL Dispase treatment
Transferring embryo regions between dishes must be done with care to ensure that they do not become stuck in the pipette or rise and burst at the meniscus [the latter occurs readily if embryos are transferred from a warm to a cold solution, e.g., Dispase solution (room temperature) to cold L-15 medium].To avoid, transfer under the microscope, avoid air bubbles, and transfer only a few embryo regions at a time(3)(4).Top tip 2: It is essential to optimise the Dispase concentration and incubation period.Tissues exposed to low concentrations of Dispase (even for longer incubation periods) will fail to separate; those exposed to high concentrations of Dispase, or incubated for too long, will become sticky, leading to a failure to obtain cleanly isolated tissue layers.Top tip 3: For mouse tissues, which are more delicate than chick, reduce time in Dispase by 20%.Top tip 4: Dispase-treated tissue fragments are transferred with a minimal volume of liquid into a large volume of L-15 medium, negating the need to inactivate the enzyme.However, if necessary, Dispase can be inactivated using a drop of 10% HINGS or BSA.